Bottom Line:
In PINK1(-/-) axons, damaged mitochondria did not accumulate Parkin and failed to be engulfed in autophagosomes.Similarly, initiation of mitophagy was blocked in Parkin(-/-) axons.Local mitophagy likely provides rapid neuroprotection against oxidative stress without a requirement for retrograde transport to the soma.

fig5: Parkin is recruited to axonal mitochondria damaged with mt-KR. (A) YFP-Parkin accumulates on a fraction of axonal mitochondria in the outlined area of mt-KR activation. (B) Line scan of the axon in A with cyan arrowheads marking two YFP-Parkin–positive mitochondria. (C and D) YFP-Parkin remained diffuse despite irradiation in the outlined area when mt-DsRed replaced mt-KR. Mitochondria with YFP-Parkin levels more than twice the background were scored as Parkin-positive here and in subsequent figures. Orange and brown arrowheads denote corresponding points in images and line scans. (E) Frequency of YFP-Parkin recruitment to irradiated mitochondria. n = 131–140 mitochondria from 12 transfections. **, P < 0.001. Error bars represent means ± SEM. AU, arbitrary unit. Bars, 5 µm.

Mentions:
In nonneuronal cells, mitochondrial depolarization causes recruitment of Parkin from the cytoplasm to the mitochondrial outer membrane where it promotes the onset of mitophagy (Narendra et al., 2008; Geisler et al., 2010). In neurons, Parkin translocation has been reported in the cell bodies of neurons treated with CCCP or valinomycin (Seibler et al., 2011; Cai et al., 2012) but was not observed in axons treated with CCCP (Van Laar et al., 2011; Cai et al., 2012). We, in contrast, had observed accumulation of Parkin on axonal mitochondria in response to global depolarization with Antimycin A (Wang et al., 2011). Because axonal Parkin recruitment might be obscured by either overexpression of YFP-Parkin in the cytosol, excessively toxic exposure to CCCP, or failure to observe the recruitment before rapid mitophagy, we reexamined the question with locally restricted and near-physiological levels of mitochondrial damage 20 min after irradiation of mitochondria expressing mt-KR and mt-BFP with 555 nm of light. When expressed at high levels, YFP-Parkin localizes to mitochondria and arrests their motility even in the absence of mitochondrial damage (Wang et al., 2011). At low levels, however, YFP-Parkin was diffuse in the axonal cytoplasm with only minor enrichment on a few mitochondria (Fig. 5, A and B). After activation of mt-KR, YFP-Parkin accumulated on 10% of the targeted mitochondria that were previously Parkin negative (Fig. 5, A, B, and E). This recruitment was caused by ROS production by mt-KR; only 3% of mitochondria were Parkin positive after equivalent irradiation of mt-DsRed (P < 0.01; Fig. 5, C–E). Thus, local damage to a small subset of axonal mitochondria is sufficient to recruit Parkin selectively to the affected population.

fig5: Parkin is recruited to axonal mitochondria damaged with mt-KR. (A) YFP-Parkin accumulates on a fraction of axonal mitochondria in the outlined area of mt-KR activation. (B) Line scan of the axon in A with cyan arrowheads marking two YFP-Parkin–positive mitochondria. (C and D) YFP-Parkin remained diffuse despite irradiation in the outlined area when mt-DsRed replaced mt-KR. Mitochondria with YFP-Parkin levels more than twice the background were scored as Parkin-positive here and in subsequent figures. Orange and brown arrowheads denote corresponding points in images and line scans. (E) Frequency of YFP-Parkin recruitment to irradiated mitochondria. n = 131–140 mitochondria from 12 transfections. **, P < 0.001. Error bars represent means ± SEM. AU, arbitrary unit. Bars, 5 µm.

Mentions:
In nonneuronal cells, mitochondrial depolarization causes recruitment of Parkin from the cytoplasm to the mitochondrial outer membrane where it promotes the onset of mitophagy (Narendra et al., 2008; Geisler et al., 2010). In neurons, Parkin translocation has been reported in the cell bodies of neurons treated with CCCP or valinomycin (Seibler et al., 2011; Cai et al., 2012) but was not observed in axons treated with CCCP (Van Laar et al., 2011; Cai et al., 2012). We, in contrast, had observed accumulation of Parkin on axonal mitochondria in response to global depolarization with Antimycin A (Wang et al., 2011). Because axonal Parkin recruitment might be obscured by either overexpression of YFP-Parkin in the cytosol, excessively toxic exposure to CCCP, or failure to observe the recruitment before rapid mitophagy, we reexamined the question with locally restricted and near-physiological levels of mitochondrial damage 20 min after irradiation of mitochondria expressing mt-KR and mt-BFP with 555 nm of light. When expressed at high levels, YFP-Parkin localizes to mitochondria and arrests their motility even in the absence of mitochondrial damage (Wang et al., 2011). At low levels, however, YFP-Parkin was diffuse in the axonal cytoplasm with only minor enrichment on a few mitochondria (Fig. 5, A and B). After activation of mt-KR, YFP-Parkin accumulated on 10% of the targeted mitochondria that were previously Parkin negative (Fig. 5, A, B, and E). This recruitment was caused by ROS production by mt-KR; only 3% of mitochondria were Parkin positive after equivalent irradiation of mt-DsRed (P < 0.01; Fig. 5, C–E). Thus, local damage to a small subset of axonal mitochondria is sufficient to recruit Parkin selectively to the affected population.

Bottom Line:
In PINK1(-/-) axons, damaged mitochondria did not accumulate Parkin and failed to be engulfed in autophagosomes.Similarly, initiation of mitophagy was blocked in Parkin(-/-) axons.Local mitophagy likely provides rapid neuroprotection against oxidative stress without a requirement for retrograde transport to the soma.